In recent years, as part of genome projects, the genomes of number of species of plants and animals have been completely sequenced. The ultimate goal of these genome projects is to identify the biological function of every gene in the genome. The functions of some of the genes have already been identified by various approaches. One approach to functional studies of gene/s is to knockout expression of target gene and monitor the effects of silencing phenotypically as well as biochemically. This is being carried out by using insertional mutagenesis, either by T-DNA or transposable elements, to knockout expression of target gene in transgenic plants. In addition to the insertional mutagenesis, overexpression of proteins is also being used to study function of genes. However, there are some drawbacks to each approach. Insertional mutagenesis results in complete inactivation of target gene, which may complicate the studies of embryo specific genes, especially if the gene is crucial for embryo development. Another point for concern is study of the gene in multi-gene family, where function of mutant gene could be compensated. In recent years, an alternative method for gene silencing to study gene function, virus induced gene silencing (VIGS), has emerged. This process facilitates targeted post-transcriptional gene silencing (PTGS). The host cell gene is being actively transcribed but no RNA accumulates or accumulates at levels lower than normal levels. This method combines new developments in virus-based expression vectors and discoveries in gene silencing. Segment(s) of host gene(s) are amplified in cells through infection with virus vectors capable of replication in plant cells. The presence of these aberrant or overexpressed RNA segments in cells results in the degradation of host mRNA containing the same sequence. It is reported that VIGS is caused by double stranded RNA formation through an RNA mediated defense mechanism. VIGS often results in a special phenotype, indicative of gene silencing. For example, inactivation of cellulose synthase gene resulted in much shorter internode length, small leaves, and a “dwarf” phenotype. Expression of mRNA fragments from phytoene desaturase using tobacco mosaic virus and potato virus X resulted in the discoloration of upper leaves of infected plant. See, U.S. Pat. No. 6,376,752. The cause of discoloration is a decline in the levels phytoene desaturase mRNA leading to lower levels of protein accumulation.
However, these prior art methods of VIGS involve the use of only mono-partite (single component) plant viral vectors with a limited host range. Further, these vectors are not capable of only local spread without systemic movement. Therefore, there is a need for developing methods involving the use of viral vectors that have a wider host-range and/or are incapable of systemic movement Further, the prior art vectors have been used for silencing of only one gene at a time. Given the number of genes whose function is not yet understood, vectors that can induce multiple gene silencing would be desirable, particularly for studies on metabolic pathways.